Nodexus? previously developed NX One system leverages node-pore sensing electrical detection (non- marker-based) and fluorescence-based marker detection of cells in combination with low-shear microfluidic valving for viable cell sorting and single-cell isolation. In this proposal, we highlight a new instrument that integrates our demonstrated node-pore sensing (?NPS?) single-cell isolation with label-free multi-marker screening for the quickly emerging cancer stem cell (CSC) space, efficient biomarker discovery and functional studies for targeted therapy development would be transformative, but easy access to viable, functionally-preserved live single-cells is not possible using any single commercial instrument. While the platform will eventually be applicable across more cancer subtypes, our initial point of entry into this sector focuses on urothelial carcinoma of the bladder (UCB). Urothelial carcinoma of the bladder is the most common malignancy of the urinary tract, with >600,000 living with bladder cancer and ~79,030 new cases and 16,870 deaths per year in the United States. From extensive conversations with experts in the field, we have found critical pain points related to UCB that the Nodexus platform can address. CSCs are a subpopulation within a heterogeneous mixture of cancer cells that have enhanced pro- malignant properties, but the contribution of specific markers to stem cell-like traits and their clinical utility as biomarkers have not been conclusively determined, making targeted therapy for these cells extremely challenging. A better understanding of the molecular mechanisms underlying urothelial CSC regulation and identification of key molecules associated with CSC generation and maintenance are pivotal for the determination of universally accepted, clinically-accurate biomarkers for early cancer detection and monitoring following transurethral resection of bladder tumor (TURBT) as well as the development of effective targeted therapies. The complexity with studying CSCs is immensely increased due to the heterogeneity within tumors and the variance in CSC-like traits and functional importance in different cancer subtypes. Conclusive confirmation of marker-associated functionality will open the door for targeted therapy development. Further studies of comprehensive panels of markers performed simultaneously will provide significant value for downstream studies for conclusive biomarker identification and optimized therapies. Critically, the functional relevance of such markers must be evaluated and understood; this requires being able to comprehensively screen for numerous markers, isolate single-cell populations to unveil masked heterogeneity, and perform downstream functional studies (e.g. growth, invasion, and resistance to chemotherapeutic agents) on these viable, functionally-preserved isolated single-cells. While existing technologies, such as FACS, MACS, and CyTOF have provided tremendous value within the broader space, none provide label-free easy access to extensively characterized, live, functionally preserved single CSCs in a manner that the Nodexus platform will offer.